Project Summary/Abstract The WHO and CDC have declared Gram negative antibiotics as one of the greatest unmet needs. Indeed, the accelerating problem of antibiotic resistance threatens up to 10 million lives/year. Despite the urgent need for new antibiotics, Gram negative organisms are challenging to target because they have impermeable membranes and efflux pumps to resist xenobiotics. Complicating matters, identifying novel natural products remains, a significant challenge: i) classic approaches that use bioactivity-guided fractionation of organism extracts are slow; ii) removed from the context of their native or symbiotic environments, microbial organisms cannot always be coaxed into producing their varied metabolites in the lab; and iii) heterologous expression in different hosts remains a limitation. In work leading up to this proposal, we have developed versatile synthetic biology platform that can overcome each of these challenges. Here, we will use this platform to produce large libraries of potential antimicrobial molecules including >10,000 natural product derivatives, >100,000 lectin variants, and >1M cyclic peptides. The output from our platform will be screened against Gram negative pathogens. A key innovation of our platform is that enzymes in a biosynthetic pathway are overexpressed lysates or made in cell-free protein synthesis to construct cell-free ?units? following a chemical engineering paradigm that can then be used to recreate the pathway or combinatorially diversify it. We have recently made key advancements in DNA sequencing workflows, microfluidics, cell-free systems, machine learning, and screening platforms to facilitate our goals. In Aim 1, we will develop a unit operation based antibiotic expression systems and generate libraries of novel compounds. In Aim 2, we will generate libraries of antimicrobial peptides in stable cyclic scaffolds. In Aim 3, we will extend our technology to generate libraries of lectins that target Gram negative pathogens. In Aim 4, which connects to all other aims, we will screen libraries from Aims 1, 2, and 3 for biological activity. We expect that our discovery-centered approach will be the first of its kind in offering high-throughput experimentation in a cell-free environment. It will uniquely (i) avoid inherent limitations of whole-cell viability, (ii) permit design-build-test (DBT) iterations without the need to reengineer organisms, and (iii) explore combinatorial and modular assembly of pathways through the use of well-defined experimental conditions that can use chemical and physical manipulations not possible in cells. This work will add new knowledge for the biosynthetic mechanisms responsible for the privileged class of natural product antibiotics and provide us with tools to systematically engineer them. Furthermore, it will deliver new chemical matter to serve as starting points for optimization and development of therapeutics.